Targeted mutagenesis in spirulina

ABSTRACT

This disclosure describes techniques for creating stable, targeted mutations in Spirulina (Athrospiria) and Spirulina having stable, targeted mutations.

PRIORITY CLAIM

This application is a continuation of U.S. application Ser. No.15/510,028, filed Mar. 9, 2017, which is a national stage application ofinternational patent application No. PCT/US15/49214, entitled “TargetedMutagenesis In Spirulina,” and filed Sep. 9, 2015, which claims priorityto U.S. Provisional Patent Application No. 62/047,811, entitled“Targeted Mutagenesis in Spirulina,” and filed Sep. 9, 2014, thecontents of each of which is incorporated in its entirety.

SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is LUBI-006/02US_SeqUst_ST25.txt. The text file isabout 73 KB, was created on Jul. 2, 2018, and is being submittedelectronically via EFS-Web.

BACKGROUND

Cyanobacteria, also called blue-green algae, are photosyntheticorganisms that use chlorophyll A and water to reduce carbon dioxide andgenerate energy-containing compounds. Spirulina are free-floating,filamentous cyanobacteria that include the species Arthrospira platensisand Arthrospira maxima. These two species were formerly classified inthe genius Spirulina, but are now classified in the genus Arthrospira.However, the term “Spirulina” remains in use.

Cyanobacteria are generally amenable to genetic manipulation. However,genetic engineering tools for Spirulina are limited. Many techniques forgenetic manipulation are based on introducing exogenous (foreign)genetic material into a bacterial cell. Cells must be in a state of“competence” to take up genetic material from the surroundingenvironment. Some types of bacteria are able to naturally take upgenetic material. These types of bacteria are referred to as having“natural competence.” More commonly, “artificial competence” is inducedby making a cell temporarily permeable to genetic material. Techniquesfor introducing artificial competence by increasing the permeability ofan outer cell membrane include incubation in chemical solutions, heatshock, and electroporation which subjects a cell to an electric field. Acell in a state of competence that uptakes exogenous genetic materialand incorporates the new genetic material into its genome is said to behave undergone “transformation.”

Spirulina has been long recognized as difficult to transform by randomintegration of DNA into a Spirulina chromosome, and applicant is notaware of any reports claiming modification of the Spirulina genome bytargeted introduction of DNA into specific, predetermined chromosomelocations. Attempts using electroporation to introduce a gene forchloramphenicol resistance have resulted in chloramphenicol resistanceunder certain electroporation conditions, but the transformation was notstable (i.e. the chloramphenicol resistance could not be sustained).Subsequent attempts to transform Spirulina with a gene forchloramphenicol resistance coupled to a strong promoter by usingelectroporation achieved cells that grew in the presence ofchloramphenicol for 12 months, but the method only allowed the gene forchloramphenicol resistance to be located at random (un-targeted)locations in a Spirulina chromosome, and even this random integration ofexogenous DNA was not conclusively demonstrated. Recently, randommutagenesis has been achieved in S. platensis (A. platensis) usingatmospheric and room-temperature plasma (ARTP). However, randommutagenesis does not transform the Spirulina cells through introducingexogenous genetic material, rather mutations are introduced at randomsites in the genome. A lack of understanding of how to stably introduceforeign DNA to predetermined chromosome locations into cyanobacteria,Spirulina in particular, is recognized as a challenge to working withcyanobacteria as compared to other organisms such as E. coli or yeast.Although there was some level of success with random mutagenesis, thisresearch also highlighted the continued lack of an effective system formutation of S. plantensis by introduction and expression an exogenousgene. Moreover, none of the techniques described above have attempted tointroduce targeted mutations to specific, pre-determined regions of theSpirulina genome.

A need still exists for a technique to efficiently create stabletransformants in Spirulina. Moreover, there is also a need fortechniques that allow for targeted introduction of mutations in theSpirulina genome.

BRIEF SUMMARY

This disclosure describes techniques for introducing stable, targetedmutations to the genome of Spirulina. This disclosure also describesSpirulina modified to contain stable, targeted mutations. Additionally,this disclosure described techniques for using Spirulina that have beenmodified to include one or more stable, targeted mutation to manufactureproducts of interest.

This disclosure describes methods of creating targeted mutations inSpirulina by contacting the Spirulina with an osmotic stabilizer,contacting the Spirulina with a vector having homology arms, andinducing artificial competence in the Spirulina. In an embodiment, theSpirulina may be Athrospiria platensis NIES-39 or Arthrospira sp. PCC8005. The osmotic stabilizer may be, but is not limited to, polyethyleneglycol (PEG), ethylene glycol, glycerol, glucose, sucrose, or anycombination thereof. The vector may be a DNA vector, a linear vector, acircular vector, a single stranded polynucleotide, or a double strandedpolynucleotide. In an embodiment the vector may be, for example, apApl-pilA/aadA plasmid.

In an embodiment the homology arms may be the same length. In anembodiment the homology arms may be different lengths. One or both ofthe homology arms may be at least about 500 bp, at least about 1000 bp,at least about 1500 bp, or at least about 2000 bp.

In an embodiment the artificial competence may be induced by any knowntechnique for introducing artificial competence in prokaryotic cellsincluding incubation in a solution containing divalent cations,electroporation, and ultrasound.

In an embodiment, the method may also include contacting the Spirulinawith a pH balancer. The Spirulina may be contacted by the pH balancerprior to or after contacting the Spirulina with the vector. In anembodiment a pH of the Spirulina after contacting with the pH balanceris about 7.0 to about 8.0.

This disclosure describes Spirulina comprising at least one stable,targeted mutation. In an embodiment the Spirulina is Athrospiriaplatensis, Athrospiria platensis NIES-39, or Arthrospira maxima. In anembodiment, the stable targeted mutation is inherited for at least 5generations, at least 10 generations, at least 20 generations, at least30 generations, at least 40 generations, or at least 50 generations. Themutation may be any type of alteration to the genome including adeletion or disruption of at least a portion of a gene, an insertion ofan additional copy of an endogenous gene, or addition of an exogenousgene. In an embodiment, the targeted mutation may include addition of anexogenous protein domain including post-translational modificationsites, protein-stabilizing domains, cellular localization signals, andprotein-protein interaction domains. In an embodiment, the targetedmutation comprises addition of a nucleic acid sequence that is nottranslated into a protein including, but not limited to, a non-codingRNA molecule, a gene regulatory element, a promoter, a regulatoryprotein binding site, a RNA binding site, a ribosome binding site, atranscriptional terminator, or a RNA-stabilizing element.

This disclosure also describes a Spirulina cell lacking at least oneprotein as a result of introducing a modification at the loci of theprotein by transformation of the Spirulina cell with at least one DNAconstruct comprising a sequence homologous with at least a portion ofthe loci and the modification and integration of the DNA construct atthe loci, the Spirulina cell being otherwise capable of functioning inits native manner.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows left and right homology arms designed to target the pilAgene locus (NIES39_C03030) in A. platensis strain NIES-39.

FIG. 2 shows a pApl-pilA/aadA plasmid used for targeted mutagenesis inA. platensis strain NIES-39. The plasmid of FIG. 2 has nucleotidesequence SEQ ID NO: 1.

FIG. 3A shows a schematic of homologous recombination at the pilA genelocus (NIES39_C03030) in A. platensis strain NIES-39 with the plasmid ofFIG. 2 containing the aminoglycoside adenyltransferase gene (aadA). FivePCR primer sites labeled A, B, C, D, E, F, and G are also shown.

FIG. 3B shows the results of PCR amplification with primers C/E, A/D,and B/C for both wild-type A. platensis and A. platensis followingtransformation with the plasmid of FIG. 2.

FIG. 3C shows the results of PCR amplification with primers F/G of thepilA gene sequence in wild-type A. platensis and lack of amplificationin A. platensis following transformation with the vector containingaadA.

FIG. 4 shows a pApl-pilA8005/aadA plasmid used for targeted mutagenesisin Arthrospira sp. PCC 8005. The plasmid of FIG. 4 has nucleotidesequence SEQ ID NO: 2.

FIG. 5A shows a schematic of homologous recombination at the pilA genelocus in Arthrospira sp. PCC 8005 with the plasmid of FIG. 4 containingthe aminoglycoside adenyltransferase gene (aadA). Three PCR primer siteslabeled H, I, and J are also shown.

FIG. 5B shows the results of PCR amplification with primers H/I and H/Jfor both wild-type Arthrospira sp. PCC 8005 and Arthrospira sp. PCC 8005following transformation with the plasmid of FIG. 4.

FIG. 6 shows a pApl-NS1/Prs-crtW-crtZ plasmid used to attempt tointroduce crtW and crtZ genes by targeted mutagenesis in A. platensisstrain NIES-39 to enable A. platensis to synthesize astaxanthin. Theplasmid of FIG. 6 has nucleotide sequence SEQ ID NO: 3.

FIG. 7 shows a pApl-NS1/aadA-cpcBA plasmid used to introduce cpcA andcpcB genes by targeted mutagenesis in A. platensis strain NIES-39 to addadditional copies of these two genes to Athrospira. This results in anAthrospira strain that overproduces C-phycocyanin. The plasmid of FIG. 7has the nucleotide sequence SEQ ID NO: 4.

FIGS. 8A-8C shows improvements in C-phycocyanin production in A.platensis strain NIES-39 transformed with the plasmid of FIG. 7 ascompared to wild-type A. platensis. FIG. 8D shows that the transformedA. platensis has a growth rate similar to wild-type A. platensis.

FIG. 9A shows a deeper blue color in a protein extraction from A.platensis strain NIES-39 transformed to overproduce C-phycocyanin and alighter blue protein extraction from wild-type A. platensis strainNIES-39.

FIG. 9B is a graph showing absorption differences between the proteinextractions from the transformed A. platensis strain NIES-39 andwild-type A. platensis strain NIES-39. Transformed A. platensis strainNIES-39 has greater absorption at 620 nm than the wild-type strain.

DETAILED DESCRIPTION Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art to which the invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, illustrative methodsand materials are described. For the purposes of the present invention,the following terms are defined below.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

Throughout this specification, unless the context requires otherwise,the words “comprise,” “comprises,” and “comprising” will be understoodto imply the inclusion of a stated step or element or group of steps orelements but not the exclusion of any other step or element or group ofsteps or elements.

As used herein, the terms “having,” “has,” “contain,” “including,”“includes,” “include,” and “have” have the same open-ended meaning as“comprising,” “comprises,” and “comprise” provided above.

By “consisting of” is meant including, and limited to, whatever followsthe phrase “consisting of.” Thus, the phrase “consisting of” indicatesthat the listed elements are required or mandatory, and that no otherelements may be present.

By “consisting essentially of” is meant including any elements listedafter the phrase, and limited to other elements that do not interferewith or contribute to the activity or action specified in the disclosurefor the listed elements. Thus, the phrase “consisting essentially of”indicates that the listed elements are required or mandatory, but thatother elements are optional and may or may not be present depending uponwhether or not they affect the activity or action of the listedelements.

By “about” is meant a quantity, level, value, number, frequency,percentage, dimension, size, amount, weight or length that varies by asmuch as 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% to a referencequantity, level, value, number, frequency, percentage, dimension, size,amount, weight, or length.

The present description uses numerical ranges to quantify certainparameters relating to the invention. It should be understood that whennumerical ranges are provided, such ranges are to be construed asproviding literal support for claim limitations that only recite thelower value of the range as well as claim limitations that only recitethe upper value of the range. For example, a disclosed numerical rangeof 10 to 100 provides literal support for a claim reciting “greater than10” (with no upper bounds) and a claim reciting “less than 100” (with nolower bounds) and provided literal support for and includes the endpoints of 10 and 100.

The present description uses specific numerical values to quantifycertain parameters relating to the invention, where the specificnumerical values are not expressly part of a numerical range. It shouldbe understood that each specific numerical value provided herein is tobe construed as providing literal support for a broad, intermediate, andnarrow range. The broad range associated with each specific numericalvalue is the numerical value plus and minus 60 percent of the numericalvalue, rounded to two significant digits. The intermediate rangeassociated with each specific numerical value is the numerical valueplus and minus 30 percent of the numerical value, rounded to twosignificant digits. The narrow range associated with each specificnumerical value is the numerical value plus and minus 15 percent of thenumerical value, rounded to two significant digits. These broad,intermediate, and narrow numerical ranges should be applied not only tothe specific values, but should also be applied to differences betweenthese specific values.

By “gene” is meant a unit of inheritance that occupies a specific locuson a chromosome and consists of transcriptional and/or translationalregulatory sequences and/or a coding region and/or non-translatedsequences (i.e., introns, 5′ and 3′ untranslated sequences) whether ornot such regulatory sequences are adjacent to coding and/or transcribedsequences. Accordingly, a gene includes, but is not necessarily limitedto, promoter sequences, terminators, translational regulatory sequencessuch as ribosome binding sites and internal ribosome entry sites,enhancers, silencers, insulators, boundary elements, replicationorigins, matrix attachment sites, and locus control regions.

The recitation “polynucleotide” or “nucleic acid” as used hereindesignates mRNA, RNA, cRNA, rRNA, cDNA, or DNA. The term typicallyrefers to polymeric form of nucleotides of at least 10 bases in length,either ribonucleotides or deoxynucleotides or a modified form of eithertype of nucleotide. The term includes single and double stranded formsof DNA and RNA.

As used herein, the term “DNA” includes a DNA molecule that has beenisolated free of total genomic DNA of a particular species. Therefore, aDNA segment encoding a polypeptide refers to a DNA segment that containsone or more coding sequences yet is substantially isolated away from, orpurified free from, total genomic DNA of the species from which the DNAsegment is obtained. Included within the terms “DNA segment” and“polynucleotide” are DNA segments and smaller fragments of suchsegments, and also recombinant vectors, including, for example,plasmids, cosmids, phagemids, phage, viruses, and the like.

With regard to polynucleotides, the term “exogenous” refers to apolynucleotide sequence that does not naturally occur in a wild-typecell or organism, but is typically introduced into the cell by molecularbiological techniques. Examples of exogenous polynucleotides includevectors, plasmids, and/or man-made nucleic acid constructs encoding adesired protein. With regard to polynucleotides, the term “endogenous”or “native” refers to naturally occurring polynucleotide sequences thatmay be found in a given wild-type cell or organism. A vector, plasmid,or other man-made construct that includes an endogenous polynucleotidesequence combined with polynucleotide sequences of the unmodified vectoretc. is, as a whole, an exogenous polynucleotide and may also bereferred to as an exogenous polynucleotide including an endogenouspolynucleotide sequence. Also, a particular polynucleotide sequence thatis isolated from a first organism and transferred to second organism bymolecular biological techniques is typically considered an “exogenous”polynucleotide with respect to the second organism.

Polynucleotides may comprise a native sequence (e.g., an endogenoussequence that encodes protein described herein) or may comprise avariant or fragment, or a biological functional equivalent of such asequence. Polynucleotide variants may contain one or more substitutions,additions, deletions and/or insertions, as further described herein,preferably such that the enzymatic activity of the encoded polypeptideis not substantially diminished relative to the unmodified or referencepolypeptide. The effect on the enzymatic activity of the encodedpolypeptide may generally be assessed as described herein and known inthe art.

As will be understood by those skilled in the art, the polynucleotidesequences of this disclosure can include genomic sequences,extra-genomic, and plasmid-encoded sequences and smaller engineered genesegments that express, or may be adapted to express, proteins,polypeptides, peptides and the like. Such segments may be naturallyisolated, or modified synthetically by the hand of man.

Polynucleotides may be single-stranded (coding or antisense) ordouble-stranded, and may be DNA (genomic, cDNA, or synthetic) or RNAmolecules. Additional coding or non-coding sequences may, but need not,be present within a polynucleotide of the present invention, and apolynucleotide may, but need not, be linked to other molecules and/orsupport materials.

By “coding sequence” is meant any nucleic acid sequence that contributesto the code for the polypeptide product of a gene. By contrast, the term“non-coding sequence” refers to any nucleic acid sequence that does notcontribute to the code for the polypeptide product of a gene.

The terms “complementary” and “complementarity” refer to polynucleotides(i.e., a sequence of nucleotides) related by the base-pairing rules. Forexample, the sequence “A-G-T,” is complementary to the sequence “T-C-A.”Complementarity may be “partial,” in which only some of the nucleicacids' bases are matched according to the base pairing rules. Or, theremay be “complete” or “total” complementarity between the nucleic acids.The degree of complementarity between nucleic acid strands hassignificant effects on the efficiency and strength of hybridizationbetween nucleic acid strands.

The percent identity of two sequences, whether nucleic acid or aminoacid sequences, is the number of exact matches between two alignedsequences divided by the length of the shorter sequences and multipliedby 100. An approximate alignment for nucleic acid sequences is providedby the Smith-Waterman algorithm. The Smith-Waterman algorithm can beapplied to amino acid sequences by using a known scoring matrix (e.g.,the scoring matrix developed by Dayhoff) and normalized by anywell-known technique such as the Gribskov method. One implementation ofthis algorithm to determine percent identity of a sequence is providedby the Genetics Computer Group (Madison, Wis.) in the “BestFit” utilityapplication. The default parameters for this method are described in theWisconsin Sequence Analysis Package Program Manual, Version 8 (1995)(available from Genetics Computer Group, Madison, Wis.). Other suitableprograms for calculating the percent identity or similarity betweensequences are generally known in the art, for example, the MPSRCHpackage of programs copyrighted by the University of Edinburgh,developed by John F. Collins and Shane S. Sturrok, and distributed byIntelliGenetics, Inc. (Mountain View, Calif.), and BLAST, used withdefault parameters. Details of these programs can be found at thefollowing internet address: http://blast.ncbi.nlm.nih.gov/Blast.cgi.

“Polypeptide,” “polypeptide fragment,” “peptide,” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues andto variants and synthetic analogues of the same. Thus, these terms applyto amino acid polymers in which one or more amino acid residues aresynthetic non-naturally occurring amino acids, such as a chemicalanalogue of a corresponding naturally occurring amino acid, as well asto naturally-occurring amino acid polymers. In certain aspects,polypeptides may include enzymatic polypeptides, or “enzymes,” whichtypically catalyze (i.e., increase the rate of) various chemicalreactions. Exemplary nucleotide sequences that encode the proteins andenzymes of the application encompass full-length referencepolynucleotides, as well as portions of the full-length or substantiallyfull-length nucleotide sequences of these genes or their transcripts orDNA copies of these transcripts. Portions of a nucleotide sequence mayencode polypeptide portions or segments that retain the biologicalactivity of the reference polypeptide.

“Transformation” refers to the stable, heritable alteration in a cellresulting from the uptake and incorporation of exogenous nucleotidesinto the host-cell genome; also, the transfer of an exogenous gene fromone organism into the genome of another organism. Exogenous nucleotidesmay include gene foreign to the target organism or addition of anucleotide sequence present in the wild-type organism.

“Targeted mutation” means a change in the DNA sequence of the genome ata pre-determined (specified) genome location. In some cases a targetedmutation will involve the introduction of a pre-determined (specified)DNA sequence alteration at the pre-determined genome location. In othercases a targeted mutation will involve the introduction of a random DNAsequence alteration at the pre-determined genome location.

“Stable” when describing the results of a genetic modification caused bytransformation refers to a genetic modification that is maintained in atleast a portion of a population of cells for ten or more generations orfor a length of time equal or greater to ten times the averagegeneration time for the modified organism.

“Competent” refers to the ability of a cell to take up extracellularnucleotides from the surrounding environment. A cell may be “naturallycompetent” or “artificially competent.” Naturally competent cells areable to take up nucleotides from their surrounding environment undernatural conditions. Artificially competent cells are made passivelypermeable to extracellular nucleotides by exposing the cell toconditions that do not normally occur naturally including incubation ina solution of divalent cations, heat shock, electroporation, andultrasound.

The terms “wild-type” and “naturally occurring” are used interchangeablyto refer to an organism, gene, or gene product that has thecharacteristics of that organism, gene or gene product (e.g., apolypeptide) when isolated from a naturally occurring source. Awild-type organism, gene, or gene product is that which is mostfrequently observed in a population and is thus arbitrarily designed the“normal” or “wild-type” form.

As used herein “Spirulina” is synonymous with “Arthrospira.” The genusArthrospria includes 57 species of which 22 are currently taxonomicallyaccepted. Thus, reference to “Spirulina” or “Arthrospira” withoutfurther designation includes reference to any of the following species:A. amethystine, A. ardissonei, A. argentina, A. balkrishnanii, A.baryana, A. boryana, A. braunii, A. breviarticulata, A. brevis, A.curta, A. desikacharyiensis, A. funiformis, A. fusiformis, A. ghannae,A. gigantean, A. gomontiana, A. gomontiana var. crassa, A. indica, A.jenneri var. platensis, A. jenneri Stizenberger, A. jenneri f. purpurea,A. joshii, A. khannae, A. laxa, A. laxissima, A. laxissima, A.leopoliensis, A. major, A. margaritae, A. massartii, A. massartii var.indica, A. maxima, A. meneghiniana, A. miniata var. constricta, A.miniata, A. miniata f. acutissima, A. neapolitana, A. nordstedtii, A.oceanica, A. okensis, A. pellucida, A. platensis, A. platensis var.non-constricta, A. platensis f. granulate, A. platensis f. minor, A.platensis var. tenuis, A. santannae, A. setchellii, A. skujae, A.spirulinoides f. tenuis, A. spirulinoides, A. subsalsa, A. subtilissima,A. tenuis, A. tenuissima, and A. versicolor.

All literature and similar materials cited in this application,including patents, patent applications, articles, books, treatises, andInternet web pages are expressly incorporated by reference in theirentirety for any purpose. When definitions of terms in incorporatedreferences appear to differ from the definitions provided in thisapplication, the definition provided in this application shall control.

Targeted Mutations in Spirulina

In an aspect of the invention, a vector having homology arms is taken upby a Spirulina cell in a state of competence and subsequently integratedinto one or more chromosomes of the cell. Homologous recombinationguided by the sequences of the homology arms changes the genome of thecell due to differences between the original nucleic acid sequence ofthe genome and the nucleic acid sequence of the vector region betweenthe homology arms.

The present disclosure describes the first technique known to theinventors for inducing competence in Spirulina. Spirulina is notnaturally competent and the techniques disclosed herein achieve atransformation where previous techniques for transforming Spirulina havefailed. The high levels of endonucleases present in Spirulina have beenpreviously thought to make transformation impossible. Electroporationhas been limited to creating competent cells for only brief periods oftime due to the tendency of Spirulina cells to lyse when subjected toelectroporation. However, transformation by electroporation in thepresence of an appropriate osmotic stabilizer achieves transformationthat was previously not possible by other techniques.

Prior to transformation, Spirulina may be cultured and washed with anosmotic stabilizer. Spirulina may be cultured in any suitable media forgrowth of cyanobacteria such as SOT medium. SOT medium includes NaHCO₃1.68 g, K₂HPO₄ 50 mg, NaNO₃ 250 mg, K₂50₄ 100 mg, NaCl 100 mg,MgSO₄.7H₂O, 20 mg, CaCl₂.2H₂O 4 mg, FeSO₄.7H₂O 1 mg, Na₂EDTA.2H₂O 8 mg,A₅ solution 0.1 mL, and distilled water 99.9 mL. A₅ solution includesH₃BO₃ 286 mg, MnSO₄.5H₂O) 217 mg, ZnSO₄.7H₂O 22.2 mg, CuSO₄.5H₂O 7.9 mg,Na₂MoO₄.2H₂O 2.1 mg, and distilled water 100 mL. Cultivation may occurwith shaking (e.g., 100-300 rpm) at a temperature higher than roomtemperature (e.g. 25-37° C.) and under continuous illumination (e.g.20-2,000, 50-500, or 100-200 μmol photon m⁻² s⁻¹). The growing cells maybe harvested when the optical density at 750 nm reaches a predeterminedthreshold (e.g., OD₇₅₀ of 0.3-2.0, 0.5-1.0, or 0.6-0.8). A volume of theharvested cells may be concentrated by centrifugation then resuspendedin a solution of pH balancer and salt. The pH balancer may be anysuitable buffer that maintains viability of Spirulina while keeping pHof the media between 6 and 9 pH, between 6.5 and 8.5 pH, or between 7and 8 pH. Suitable pH balancers include HEPES, HEPES-NaOH, sodium orpotassium phosphate buffer, and TES. The salt solution may be NaCl at aconcentration of between 50 mM and 500 mM, between 100 mM and 400 mM, orbetween 200 mM and 300 mM. In an embodiment between 1-50 mL of 1-100 mMpH balance may be used to neutralize the pH.

Cells collected by centrifugation may be washed with an osmoticstabilizer and optionally a salt solution (e.g. 1-50 mL of 0.1-100 mMNaCl). Any amount of the culture may be concentrated by centrifugation.In an embodiment between 5-500 mL of the culture may be centrifuged. Theosmotic stabilizer may be any type of osmotic balancer that stabilizescell integrity of Spriulina during electroporation. In an embodiment,the osmotic stabilizer may be a sugar (e.g. w/v 0.1-25%) such as glucoseor sucrose. In an embodiment the osmotic stabilizer may be a simplepolyol (e.g. v/v 1-25%) including glycerine, glycerin, or glycerol. Inan embodiment the osmotic stabilizer may be a polyether including (e.g.w/v 0.1-20%) polyethylene glycol (PEG), poly(oxyethylene), orpoly(ethylene oxide) (PEO). The PEG or PEO may have any molecular weightfrom 200 to 10,000, from 1000 to 6000, or from 2000 to 4000. In anembodiment the pH balancer or buffer may be used instead of or inaddition to the osmotic stabilizer.

The present disclosure also describes creation of targeted mutations inSpirulina through homologous recombination by introduction of a vectorto competent cells. Artificial competency may be created by theelectroporation technique described above or by any other known orfuture technique for creating competency in Spirulina. Known techniquesfor introducing artificial competence in Spirulina includeelectroporation (with or without an osmotic stabilizer and with orwithout a pH balancer), incubation in a solution containing divalentcations, and ultrasound. The Spirulina cells are contacted by a vectorwhen artificial competency is induced. For example, a vector may bemixed with a solution of Spirulina cells prior to electroporation.

Electroporation may be performed in a 0.1-, 0.2- or 0.4-cmelectroporation cuvette at between 0.6 and 10 kV/cm, between 2.5 and 6.5kV/cm, or between 4.0 and 5.0 kV/cm; between 1 and 100 μF, between 30and 70 μF, or between 45 and 55 μF; and between 10 and 500 mΩ, between50 and 250 mΩ, or between 90 and 110 mΩ. In an embodimentelectroporation may be performed at 4.5 kV/cm, 50 μF, and 100 mΩ.

Following electroporation the cells may be grown in the presence of oneor more antibiotics selected based on resistance conferred throughsuccessful transformation with the plasmid. Post-electroporationculturing may be performed at reduced illumination levels (e.g. 5-500,10-100, or 30-60 μmol photon m⁻² s⁻¹). The culturing may also beperformed with shaking (e.g. 100-300 rpm). The level of antibiotics inthe media may be between 5 and 100 μg/mL. Post-electroporation culturingmay be continued for 1-5 days or longer. Successful transformantsidentified by antibiotic resistance may be selected over a time courseof 1 week to 1 month on plates or in 5-100 mL of SOT medium supplementedwith 0.1-2.0 μg of appropriate antibiotics.

By “vector” is meant a polynucleotide molecule, preferably a DNAmolecule derived, for example, from a plasmid, bacteriophage, yeast, orvirus, into which a polynucleotide can be inserted or cloned. A vectormay contain one or more synthetic nucleotides or nucleic acid analogues.A vector may contain one or more unique restriction sites. The vectorcan be one which, when introduced into the host cell, is integrated intothe genome and replicated together with the chromosome(s) into which ithas been integrated. Such a vector may comprise one or more specificsequences that allow recombination into a particular, desired site ofthe host chromosome. These specific sequences may be homologous tosequences present in the wild-type genome. A vector system can comprisea single vector or plasmid, two or more vectors or plasmids, some ofwhich increase the efficiency of targeted mutagenesis, or atransposition. The choice of the vector will typically depend on thecompatibility of the vector with the host cell into which the vector isto be introduced. The vector can include a reporter gene, such as agreen fluorescent protein (GFP), which can be either fused in frame toone or more of the encoded polypeptides, or expressed separately. Thevector can also include a positive selection marker such as anantibiotic resistance gene that can be used for selection of suitabletransformants. The vector can also include a negative selection markersuch as the type II thioesterase (tesA) gene or the Bacillus subtilisstructural gene (sacB). Use of a reporter or marker allows foridentification of those cells that have been successfully transformedwith the vector.

In an embodiment, the vector includes one or two homology arms that arehomologous to DNA sequences of the Spirulina genome which are adjacentto the targeted locus. The sequence of the homology arms may beidentical or similar to the regions of the Spirulina genome to which thehomology arms are complementary. “Homology” or “homologous” as usedherein includes both homologous, identical sequences and homologous,non-identical sequences. Homologous non-identical sequences refer to afirst sequence which shares a degree of sequence identity with a secondsequence, but whose sequence is not identical to that of the secondsequence. For example, a polynucleotide comprising the wild-typesequence of a mutant gene is homologous and non-identical to thesequence of the mutant gene. As used herein, the degree of homologybetween the two homologous, non-identical sequences is sufficient toallow homologous recombination there between, utilizing normal cellularmechanisms. Two homologous non-identical sequences can be any length andtheir degree of non-homology can be as small as a single nucleotide(e.g., for a genomic point mutation introduced targeted homologousrecombination) or as large as 10 or more kilobases (e.g., for insertionof a gene at a predetermined locus in a chromosome). Two polynucleotidescomprising homologous non-identical sequences need not be the samelength. For example, an exogenous polynucleotide (i.e., vectorpolynucleotide) of between 20 and 10,000 nucleotides or nucleotide pairscan be used.

The characterization of two sequences as homologous, identical sequencesor homologous, non-identical sequences may be determined by comparingthe percent identity between the two sequences (polynucleotide or aminoacid). Homologous, identical sequences have 100% sequence identity.Homologous, non-identical sequences may have sequence identity greaterthan 80%, greater than 85%, greater than 90%, greater than 91%, greaterthan 92%, greater than 93%, greater than 94%, greater than 95%, greaterthan 96%, greater than 97%, greater than 98%, or greater than 99%.

The homology arms may be any length that allows for site-specifichomologous recombination. A homology arm may be any length between about2000 bp and 500 bp including all integer values between. For example, ahomology arm may be about 2000 bp, about 1500 bp, about 1000 bp, orabout 500 bp. In embodiments having two homology arms the homology armsmay be the same or different length. Thus, each of the two homology armsmay be any length between about 2000 bp and 500 bp including all integervalues between. For example each of the two homology arms may be about2000 bp, about 1500 bp, about 1000 bp, or about 500 bp.

A portion of the vector adjacent to one or both (i.e., between) homologyarms modifies the targeted locus in the Spirulina genome by homologousrecombination. Techniques for homologous recombination in otherorganisms are generally known (see, e.g., Kriegler, 1990, Gene transferand expression: a laboratory manual, Stockton Press). The modificationmay change a length of the targeted locus including a deletion ofnucleotides or addition of nucleotides. The addition or deletion may beof any length. The modification may also change a sequence of thenucleotides in the targeted locus without changing the length. Thetargeted locus may be any portion of the Spirulina genome includingcoding regions, non-coding regions, and regulatory sequences. In anembodiment the mutation may delete a gene thereby creating a knock-outorganism. In an embodiment the mutation may add a gene that functions asa reporter or marker (e.g., GFP or antibiotic resistance). In anembodiment the mutation may add an exogenous gene. In an embodiment themutation may add an endogenous gene under control of an exogenouspromoter (e.g., strong promoter, inducible promoter, etc.).

A vector for use in the targeted mutagenesis described above may beproduced by assembling a vector backbone with an insert sequence. Thevector may be created by any known or later developed techniqueincluding restriction enzyme digest followed by ligation or Gibsonassembly. Gibson assembly may be performed by combining DNA sequencesthat will become portions of vector backbone with an exonuclease, DNApolymerase, and DNA ligase then incubating at 50° C. for one hour. Thevector backbone may be selected for compatibility with the targetorganism. For Spirulina, suitable vector backbones include, but are notlimited to DNA plasmids. The vector backbone may be converted from acontinuous loop to a linear form by treatment with an appropriaterestriction endonuclease. The ends thereby formed are treated withalkaline phosphatase to remove 5′-phosphate end groups so that thevector may not reform a continuous loop in a DNA ligase reaction withoutfirst incorporating an insert segment.

The insert sequence includes one or two homology arms and a nucleotidesequence that, due to differences between the nucleotide sequences ofthe insert and the wild-type genome sequence of the Spirulina, modify alocus of the Spirulina. The insert sequence includes the one or twoflanking regions adjacent to the locus of interest that correspond tothe homology arms which are homologous to regions of the Spirulinagenome and a portion that is different from the Spirulina genome. Theportion of the insert sequence that differs from the wild-typenucleotide sequence of the Spirulina leads to modification of theSpirulina genome due to those differences. The modification may include,but is not limited to, a point mutation, addition of a gene, addition ofa regulatory element, addition of a coding region, addition of anon-coding region, deletion of a gene, deletion of a portion of a gene,or deletion of a regulatory element. Generally, it is well-known thatstrong E. coli promoters work well in Cyanobacteria. If the sequence ofthe target organism is known, a published sequence may be used to designthe insert sequence with homology arm(s). Several strains of Spirulinahave been sequenced such as Athrospira plantensis NIES-39. If thesequence is not known a portion of the genome containing the loci ofinterest may be amplified using PCR and suitable primers. This regionmay be subsequently sequenced using techniques known to one skilled inthe art. In an embodiment an amplified region of the genome may be usedto carry out overlap extension PCR to create a deletion. In anembodiment an amplified region of the genome may be digested with arestriction endonuclease that cuts the nucleotide sequence in one placeand ligated to an insert nucleotide sequence that has be prepared withcompatible ends.

The insert sequence is digested with one or more restrictionendonucleases to create ends that are compatible with the ends of thevector backbone. Alternatively, short sequences (e.g., 16-25 bp) thatare identical to the terminal regions of the vector backbone are addedto the ends of the insert sequence, and these two fragments arecircularized by the Gibson assembly method. The length of the insertsequence will be the length of the modifications to the loci of interestand the length of the flanking regions. For example, if the length ofthe modified sequence is 500 bp and two homology arms each of 2000 bpare desired, then then the entire length of the sequence will be 4500bp. The polynucleotides sequences used as vectors in the presentinvention, regardless of the length of the coding sequence itself, maybe combined with other sequences, such as promoters, transcriptionalterminators, additional restriction enzyme sites, multiple cloningsites, other coding segments, and the like, such that their overalllength may vary considerably. It is therefore contemplated that apolynucleotide fragment of almost any length may be employed, with thetotal length preferably being limited only by the ease of preparationand use in the intended recombinant nucleotide protocol.

Illustrative Applications Neutral Lipid (Wax Ester and Triglyceride)Production

In an embodiment Spirulina may be modified to increase production ofneutral lipids, including wax esters and/or triglycerides, beyond thelevel produced by a wild-type Spirulina grown under the same condition.Triglycerides and wax esters may be used as a feedstock in theproduction of biofuels and/or various specialty chemicals. For example,triglycerides may be subject to a transesterification reaction, in whichan alcohol reacts with triglyceride oils, such as those contained invegetable oils, animal fats, recycled greases, to produce biodieselssuch as fatty acid alkyl esters. Such reactions also produce glycerin asa by-product, which can be purified for use in the pharmaceutical andcosmetic industries. Triglycerides, or triacylglycerols (TAG), consistprimarily of glycerol esterified with three fatty acids, and yield moreenergy upon oxidation than either carbohydrates or proteins.Triglycerides provide an important mechanism of energy storage for mosteukaryotic organisms. In mammals, TAGs are synthesized and stored inseveral cell types, including adipocytes and hepatocytes. In contrast toeukaryotes, the observation of triglyceride production in prokaryoteshas been limited to certain actinomycetes, such as members of the generaMycobacterium, Nocardia, Rhodococcus, and Streptomyces, in addition tocertain members of the genus Acinetobacter.

Certain organisms can be utilized as a source of neutral lipids in theproduction of biofuels. For example, eukaryotic algae naturally producetriglycerides as energy storage molecules, and certain biofuel-relatedtechnologies are presently focused on the use of algae as a feedstockfor biofuels. Algae are photosynthetic organisms, and the use oftriglyceride-producing organisms such as algae provides the ability toproduce biodiesel from sunlight, water, CO₂, macronutrients, andmicronutrients.

Like algae, cyanobacteria species including Spirulina may obtain energyfrom photosynthesis, utilizing chlorophyll A and water to reduce CO₂.However, Spirulina lacks the essential enzymes involved in neutral lipidsynthesis. Addition of exogenous genes to cyanobacteria increases lipidproduction. The techniques described above may be used to add genes toSpirulina through targeted, homologous recombination. Thus, thetechniques described in this disclosure provide a way to add genes knownto increase triglyceride and wax ester production in cyanobacteria suchas genes encoding diacylglycerol acyltransferase (DGAT), phosphatidatephosphatase, acetyl-CoA carboxylase (ACCase), aldehyde forming acyl-ACPreductase (AAR), alcohol forming fatty acyl-CoA reductase and alcoholforming fatty acyl-ACP reductase (FAR). The techniques described abovemay also be used to add portions of genes or polynucleotide sequencesencoding polypeptide sequences that differ from wild-type DGAT,phosphatidate phosphatase, and/or ACCase polynucleotide sequences butthat still have DGAT enzymatic activity, phosphatidate phosphataseenzymatic activity, and/or ACCase enzymatic activity.

Acyl ACP Reductases (AARs) catalyze the reduction of acyl-ACP's to acylaldehydes, also known as fatty aldehydes. These enzymes are also knownas Fatty Acyl ACP Reductasts (FARs). Fatty aldehydes can serve as asubstrate for fatty alcohol biosynthesis by a FAR or long chain alcoholdehydrogenase (ADH). One example of an acyl-ACP reductase isPCC7942_orf1594 from S. elongatus. The family of AAR genes incyanobacteria (or FAR genes) have been identified using hidden Markovmodel protein family patterns TIGR045058 (aldehyde forming long chainfatty acyl ACP reductase) and TIGR04059 (long chain fatty aldehydedecarbonylase) from the TIGRRAMs database.

According to one non-limiting theory, certain embodiments may employAARs in conjuction with FARs or ADHs to increase synthesis of fattyalcohols, which can then be incorporated into wax esters, mainly by theDGAT-expressing (and thus wax ester-producing) photosyntheticmicroorganisms described herein. Hence, AARs can be used in any of theembodiments described herein, such as those that produce increasedlevels of free fatty alcohols, where it is desirable to turn these intowax esters. As noted above, these free fatty alcohols can then beesterified to fatty acids (in the form of acyl-ACP) by DGATs to generatewax esters.

Certain embodiments relate to the use of overexpressed AARs to increasesynthesis of fatty alcohols, and thereby increase production of waxesters in a wax ester-producing strain (e.g., a DGAT-expressing strain).For instance, certain embodiments may utilize an AAR, in combinationwith a fatty acyl reductase, and a DGAT. These embodiments may thenfurther utilize an ACP, an ACCase, or both, and/or any of themodifications to glycogen production and storage or glycogen breakdowndescribed herein.

Fatty Acyl Reductases (FAR) catalyze the two step reduction ofacyl-ACP's or acyl-COA's to acyl alcohols, also known as fatty alcohols.The first step proceeds via an acyl aldehyde intermediate, which is thenconverted in a second step to a fatty alcohol. These same enzymes canalso directly reduce fatty aldehydes to fatty alcohols (i.e. step twoonly). In this case they are sometimes referred to as fatty aldehydereductases. Fatty alcohols can serve as a substrate for wax esterbiosynthesis by a DGAT. Many fatty acyl reductases are characterized bythree conserved sequence elements. There is an NADPH binding motif, amotif characteristic of the catalytic site of NADP-utilizing enzymes,and a conserved C-terminal domain, referred to as the Male Sterile 2domain, that is of unknown function.

According to one non-limiting theory, certain embodiments may employfatty acyl reductases to increase synthesis of fatty alcohols, which canthen be incorporated into wax esters, mainly by the DGAT-expressing (andthus wax ester-producing) photosynthetic microorganisms describedherein. Hence, fatty acyl reductases can be used in any of theembodiments described herein, such as those that produce increasedlevels of free fatty alcohols, where it is desirable to turn these intowax esters. As noted above, these free fatty alcohols can then beesterified to fatty acids (in the form of acyl-ACP) by DGATs to generatewax esters.

Certain embodiments relate to the use of overexpressed fatty acylreductases to increase synthesis of fatty alcohols, and thereby increaseproduction of wax esters in a wax ester-producing strain (e.g., aDGAT-expressing strain). For instance, certain embodiments may utilize afatty acyl reductase, possibly in combination with an acyl-ACPreductase, and a DGAT. These embodiments may then further utilize anACP, an ACCase, or both, and/or any of the modifications to glycogenproduction and storage or glycogen breakdown described herein.

Diacylglycerol acyltransferases (DGATs) are members of theO-acyltransferase superfamily, which esterify either sterols ordiacylglycerols in an oleoyl-CoA-dependent manner. DGAT in particularesterifies diacylglycerols, and this esterification represents the finalenzymatic step in the production of triacylglycerols in plants, fungi,and mammals. Specifically, DGAT is responsible for transferring an acylgroup from acyl-coenzyme-A to the sn-3 position of 1,2-diacylglycerol(DAG) to form triacylglycerol (TAG). The DGAT may be an Acinetobacterbaylii ADP1 diacylglycerol acyltransferase (AtfA), a Streptomycescoelicolor DGAT, Plesiomonas Shigelloides DGAT or Alcanivoraxborkumensis DGAT. Thus, in an embodiment the DGAT protein comprises asequence set forth in any one of SEQ ID NOs: 5, 6, or 7. SEQ ID NO: 5 isthe sequence of Acinetobacter DGAT. SEQ ID NO: 6: is the sequence of aStreptomyces coelicolor DGAT. SEQ ID NO: 7: is the sequence ofAlcanivorax borkumensis DGAT. In plants and fungi, DGAT is associatedwith the membrane and lipid body fractions. In catalyzing TAGs, DGATcontributes mainly to the storage of carbon used as energy reserves. Inanimals, however, the role of DGAT is more complex. DGAT not only playsa role in lipoprotein assembly and the regulation of plasmatriacylglycerol concentration, but participates as well in theregulation of diacylglycerol levels (Biochemistry of Lipids,Lipoproteins and Membranes 171-203). DGAT proteins may utilize a varietyof acyl substrates in a host cell, including fatty acyl-CoA and fattyacyl-ACP molecules. In addition, the acyl substrates acted upon by DGATenzymes may have varying carbon chain lengths and degrees of saturation,although DGAT may demonstrate preferential activity towards certainmolecules.

A “phosphatidate phosphatase” gene as used herein includes anypolynucleotide sequence encoding amino acids, such as protein,polypeptide or peptide, obtainable from any cell source, whichdemonstrates the ability to catalyze the dephosphorylation ofphosphatidate (PtdOH) under enzyme reactive conditions, yieldingdiacylglycerol (DAG) and inorganic phosphate, and further includes anynaturally-occurring or non-naturally occurring variants of aphosphatidate phosphatase sequence having such ability. Examplephosphatidate phosphatase genes include, but are not limited to, yeastphosphatidate phosphatase, including Saccharomyces cerevisiaephosphatidate phosphatase (yPah1). The Pah1-encoded PAP1 enzyme is foundin the cytosolic and membrane fractions of the cell, and its associationwith the membrane is peripheral in nature. As expected from the multipleforms of PAP1 that have been purified from yeast, pah1Δ mutants stillcontain PAP1 activity, indicating the presence of an additional gene orgenes encoding enzymes having PAP1 activity. In an embodiment thephosphatidate phosphatase gene (Pah1) comprises a sequence set forth inSEQ ID NO: 8.

As used herein, an “acetyl CoA carboxylase” gene includes anypolynucleotide sequence encoding amino acids, such as protein,polypeptide or peptide, obtainable from any cell source, whichdemonstrates the ability to catalyze the carboxylation of acetyl-CoA toproduce malonyl-CoA under enzyme reactive conditions, and furtherincludes any naturally-occurring or non-naturally occurring variants ofan acetyl CoA carboxylase sequence having such ability. Acetyl-CoAcarboxylase (ACCase) is a biotin-dependent enzyme that catalyzes theirreversible carboxylation of acetyl-CoA to produce malonyl-CoA throughits two catalytic activities, biotin carboxylase (BC) andcarboxyltransferase (CT). The biotin carboxylase (BC) domain catalyzesthe first step of the reaction: the carboxylation of the biotinprosthetic group that is covalently linked to the biotin carboxylcarrier protein (BCCP) domain. In the second step of the reaction, thecarboxyltransferase (CT) domain catalyzes the transfer of the carboxylgroup from (carboxy) biotin to acetyl-CoA. Formation of malonyl-CoA byacetyl-CoA carboxylase (ACCase) represents the commitment step for fattyacid synthesis, because malonyl-CoA has no metabolic role other thanserving as a precursor to fatty acids. Because of this reason,acetyl-CoA carboxylase represents a pivotal enzyme in the synthesis offatty acids. The ACCase may be a Saccharomyces cerevisiae acetyl-CoAcarboxylase (γACC1), a Triticum aestivum ACCase, or a Synechococcus sp.PCC 7002 ACCAse. In an embodiment the ACCase gene comprises a sequenceset forth in SEQ ID NO: 9.

Specifically, phosphatidate phosphatase enzymes catalyze the productionof diacylglycerol molecules, an immediate pre-cursor to triglycerides,and DGAT enzymes catalyze the final step of triglyceride synthesis byconverting the diacylglycerol precursors to triglycerides. Increasedintracellular ACCase activity contributes to the increased production offatty acids because this enzyme catalyzes the “commitment step” of fattyacid synthesis. Specifically, ACCase catalyzes the production of a fattyacid synthesis precursor molecule, malonyl-CoA.

The genes or nucleotides encoding polypeptides with the desiredenzymatic activity may be introduced to the Spirulina genome by a vectorwith a nucleotide sequence encoding DGAT, phosphatidate phosphatase,ACCase, or a polynucleotide sequence that has DGAT enzymatic activity,phosphatidate phosphatase enzymatic activity, or ACCase enzymaticactivity between homology arms which target a specific locus of theSpirulina genome. The vector may be any vector suitable fortransformation of Spirulina including the vectors described above. Thepolynucleotides of the vector may be codon-optimized for expression inSpirulina. The vector may include a promoter associated with the addedgene. The promoter may be an inducible promoter. As will be understoodby those of skill in the art, it may be advantageous in some instancesto produce polypeptide-encoding nucleotide sequences possessingnon-naturally occurring codons. For example, codons preferred by aparticular prokaryotic or eukaryotic host can be selected to increasethe rate of protein expression or to produce a recombinant RNAtranscript having desirable properties, such as a half-life which islonger than that of a transcript generated from the naturally occurringsequence. Such nucleotides are typically referred to as“codon-optimized.”

In an embodiment the specific locus is be a gene that is replaced withthe nucleotide sequence of the vector. Regulatory elements such as apromoter associated with the replaced gene may be used to directtranscription of the nucleotide sequence from the vector. In anembodiment the specific locus may be a non-coding region of theSpirulina genome. The nucleotide sequence of the vector between thehomology arms may include regulatory elements such as a promoter thatare introduced into the Spirulina genome together with the genes ornucleotides encoding polypeptides with the desired enzymatic activity.Since wild-type Spirulina do not typically encode the enzymes necessaryfor triglyceride synthesis the techniques described in this disclosureprovide a way to add exogenous genes having DGAT activity, phosphatidatephosphatase activity, and/or ACCase enzymatic activity to Spirulina inorder to increase lipid production.

Reduced Glycogen Production

In an embodiment Spirulina may be modified to accumulate a reducedamount of glycogen as compared to a wild-type Spirulina grown under thesame conditions. By reducing the amount of glycogen produced, carbonassimilated by Cyanobacteria is directed to the synthesis of othercarbon-based products such as lipids and/or fatty acids. Deletion ofglycogen biosynthesis genes in cyanobacteria decrease glycogenaccumulation.

By blocking, disrupting, or down-regulating the natural glycogensynthesis and storage pathway, e.g., by gene mutation or deletion, inCyanobacteria the resulting strains of photosynthetic microorganismsincrease carbon flow into other biosynthetic pathways. Examples of otherbiosynthetic pathways include existing pathways, such as existing lipidbiosynthetic pathways, or pathways that are introduced through geneticengineering, such as fatty acid or triglyceride biosynthesis pathways.This modification of deleting a gene associated with glycogen synthesismay be combined with the modification described above to add exogenousgenes associated triglyceride synthesis. The techniques described abovemay be used to delete all or a portion of one or more genes associatedwith the glycogen synthesis and/or storage pathway in Spirulinaincluding glucose-1-phosphate adenyltransferase (g/gC) genes,phosphoglucomutase (pgm) genes, or a glycogen synthase (glgA) genes.Deletions of a portion of a glucose-1-phosphate adenyltransferase (g/gC)gene, a phosphoglucomutase (pgm) gene, or a glycogen synthase (glgA)gene that renders the resulting polypeptide without or with a reducedlevel of enzymatic activity are also contemplated. Decreased glycogensynthesis and/or accumulation may be more pronounced in Spriulina grownunder stress conditions such as reduced nitrogen.

Glycogen is a polysaccharide of glucose, which functions as a means ofcarbon and energy storage in most cells, including animal and bacterialcells. More specifically, glycogen is a very large branched glucosehomopolymer containing about 90% α-1,4-glucosidic linkages and 10% α-1,6linkages. For bacteria in particular, the biosynthesis and storage ofglycogen in the form of α-1,4-polyglucans represents an importantstrategy to cope with transient starvation conditions in theenvironment.

Glycogen biosynthesis involves the action of several enzymes. Forinstance, bacterial glycogen biosynthesis occurs generally through thefollowing general steps: (1) formation of glucose-1-phosphate, catalyzedby phosphoglucomutase (Pgm), followed by (2) ADP-glucose synthesis fromATP and glucose 1-phosphate, catalyzed by glucose-1-phosphateadenylyltransferase (GlgC), followed by (3) transfer of the glucosylmoiety from ADP-glucose to a pre-existing α-1,4 glucan primer, catalyzedby glycogen synthase (GlgA). This latter step of glycogen synthesistypically occurs by utilizing ADP-glucose as the glucosyl donor forelongation of the α-1,4-glucosidic chain.

In bacteria, the main regulatory step in glycogen synthesis takes placeat the level of ADP-glucose synthesis, or step (2) above, the reactioncatalyzed by glucose-1-phosphate adenylyltransferase (GlgC), also knownas ADP-glucose pyrophosphorylase. In contrast, the main regulatory stepin mammalian glycogen synthesis occurs at the level of glycogensynthase. As shown herein, by altering the regulatory and/or otheractive components in the glycogen synthesis pathway of photosyntheticmicroorganisms such as Cyanobacteria, and thereby reducing thebiosynthesis and storage of glycogen, the carbon that would haveotherwise been stored as glycogen can be utilized to synthesize othercarbon-based storage molecules, such as lipids, fatty acids, andtriglycerides.

In an embodiment, a Spirulina, expresses a reduced amount of thephosphoglucomutase gene. In particular embodiments, it may comprise amutation or deletion in the phosphoglucomutase gene, including any ofits regulatory elements (e.g., promoters, enhancers, transcriptionfactors, positive or negative regulatory proteins, etc.).Phosphoglucomutase (Pgm), encoded by the gene pgm, catalyzes thereversible transformation of glucose 1-phosphate into glucose6-phosphate, typically via the enzyme-bound intermediate, glucose1,6-biphosphate. Although this reaction is reversible, the formation ofglucose-6-phosphate is markedly favored.

In an embodiment, a modified Spirulina expresses a reduced amount of aglucose-1-phosphate adenylyltransferase (glgC) gene. In certainembodiments, it may comprise a mutation or deletion in the glgC gene,including any of its regulatory elements. The enzyme encoded by the glgCgene (e.g., EC 2.7.7.27) participates generally in starch, glycogen andsucrose metabolism by catalyzing the following chemical reaction:

ATP+alpha-D-glucose 1-phosphate→diphosphate+ADP-glucose Thus, the twosubstrates of this enzyme are ATP and alpha-D-glucose 1-phosphate,whereas its two products are diphosphate and ADP-glucose. TheglgC-encoded enzyme catalyzes the first committed and rate-limiting stepin starch biosynthesis in plants and glycogen biosynthesis in bacteria.It is the enzymatic site for regulation of storage polysaccharideaccumulation in plants and bacteria, being allosterically activated orinhibited by metabolites of energy flux.

The enzyme encoded by the glgC gene belongs to a family of transferases,specifically those transferases that transfer phosphorus-containingnucleotide groups (i.e., nucleotidyl-transferases). The systematic nameof this enzyme class is typically referred to asATP:alpha-D-glucose-1-phosphate adenylyltransferase. Other names incommon use include ADP glucose pyrophosphorylase, glucose 1-phosphateadenylyltransferase, adenosine diphosphate glucose pyrophosphorylase,adenosine diphosphoglucose pyrophosphorylase, ADP-glucosepyrophosphorylase, ADP-glucose synthase, ADP-glucose synthetase, ADPGpyrophosphorylase, and ADP:alpha-D-glucose-1-phosphateadenylyltransferase.

In an embodiment, Spirulina expresses a reduced amount of a glycogensynthase gene. In particular embodiments, it may comprise a deletion ormutation in the glycogen synthase gene, including any of its regulatoryelements. Glycogen synthase (GlgA), also known as UDP-glucose-glycogenglucosyltransferase, is a glycosyltransferase enzyme that catalyses thereaction of UDP-glucose and (1,4-α-D-glucosyl)_(n) to yield UDP and(1,4-α-D-glucosyl)_(n+1). Glycogen synthase is an α-retainingglucosyltransferase that uses ADP-glucose to incorporate additionalglucose monomers onto the growing glycogen polymer. Essentially, GlgAcatalyzes the final step of converting excess glucose residues one byone into a polymeric chain for storage as glycogen.

Classically, glycogen synthases, or α-1,4-glucan synthases, have beendivided into two families, animal/fungal glycogen synthases andbacterial/plant starch synthases, according to differences in sequence,sugar donor specificity and regulatory mechanisms. However, detailedsequence analysis, predicted secondary structure comparisons, andthreading analysis show that these two families are structurally relatedand that some domains of animal/fungal synthases were acquired to meetthe particular regulatory requirements of those cell types.

Crystal structures have been established for certain bacterial glycogensynthases. These structures show that reported glycogen synthase foldsinto two Rossmann-fold domains organized as in glycogen phosphorlyaseand other glycosyltransferases of the glycosyltransferases superfamily,with a deep fissure between both domains that includes the catalyticcenter. The core of the N-terminal domain of this glycogen synthaseconsists of a nine-stranded, predominantly parallel, central β-sheetflanked on both sides by seven α-helices. The C-terminal domain(residues 271-456) shows a similar fold with a six-stranded parallelβ-sheet and nine α-helices. The last α-helix of this domain undergoes akink at position 457-460, with the final 17 residues of the protein(461-477) crossing over to the N-terminal domain and continuing asα-helix, a typical feature of glycosyltransferase enzymes.

These structures also show that the overall fold and the active sitearchitecture of glycogen synthase are remarkably similar to those ofglycogen phosphorylase, the latter playing a central role in themobilization of carbohydrate reserves, indicating a common catalyticmechanism and comparable substrate-binding properties. In contrast toglycogen phosphorylase, however, glycogen synthase has a much widercatalytic cleft, which is predicted to undergo an important interdomain‘closure’ movement during the catalytic cycle.

Crystal structures have been established for certain GlgA enzymes. Thesestudies show that the N-terminal catalytic domain of GlgA resembles adinucleotide-binding Rossmann fold and the C-terminal domain adopts aleft-handed parallel beta helix that is involved in cooperativeallosteric regulation and a unique oligomerization. Also, communicationbetween the regulator-binding sites and the active site involves severaldistinct regions of the enzyme, including the N-terminus, theglucose-1-phosphate-binding site, and the ATP-binding site.

The glucose-1-phosphate adenyltransferase (g/gC), phosphoglucomutase(pgm), and/or glycogen synthase (glgA) genes may be deleted in whole orpart using a vector with homology arms that target the upstream anddownstream flanking regions of a given gene and recombines with theSpirulina genome to remove nucleotides in the region between thehomology arms or replace nucleotides with a different sequence such as areporter or marker gene. Given the presence of a reporter or marker inthe vector sequence, such as a drug-selectable marker, Spirulina cellscontaining the gene deletion can be readily isolated, identified, andcharacterized. As one example, selection and isolation may include theuse of antibiotic resistant markers known in the art (e.g., kanamycin,spectinomycin, and streptomycin). Such selectable vector-basedrecombination methods need not be limited to targeting upstream anddownstream flanking regions, but may also be targeted to internalsequences within a given gene, as long as that gene is rendered“non-functional.” The deletion of all or part of genes that participatein glycogen synthesis does not harm growth of Spriulina but reducesproduction of glycogen.

Modified Carotenoid Production

Carotenoids can be produced from fats and other basic organic metabolicbuilding blocks by Cyanobacteria including Spirulina. Carotenoidsfunction in photosynthesis to protect other components of photosyntheticsystems from oxidative stress. Carotenoids may also provide variousyellow to red shades of pigmentation. Carotenoid biosynthesis inCyanobacteria can be modified by reducing the expression of certaingenes, increasing the expression of certain genes, and/or introducingexogenous genes.

Cyanobacteria which contains deletions of crtG (2,2′-β-carotenehydroxylase) leads to increased synthesis and accumulation of zeaxanthinwhile maintaining typical rates of exponential growth. Additionally, anexpression of crtR (3,3′-β-carotene hydroxylase) or crtZ(cartonenoid-3,3′-hydroxylase) has been demonstrated to lead toproduction of zeaxanthin in the E. coli strain, where a series of genesinvolved in β-carotene synthesis are introduced, crtE, crtB, crtI andcrtY. In an embodiment the crtR gene comprises a sequence set forth inSEQ ID NO: 10. In an embodiment the crtZ gene is from Brevundimonas sp.SD212 and comprises a sequence set forth in SEQ ID NO: 11.

Additionally, introduction of crtW (β-carotene oxygenase) and crtZ(cartonenoid-3,3′-hydroxylase) leads to synthesis and accumulation ofastaxanthin and canthaxanthin in Cyanobacteria. In an embodiment thecrtW gene is from Brevundimonas sp. SD212 and comprises a sequence setforth in SEQ ID NO: 12.

The techniques described in this disclosure may be used to knock out orrender non-functional Spirulina genes in for crtG. Additionally thetechniques described in this disclosure may be used to add crtR, crtZ,and crtW genes to the Spirulina genome under control of a nativeSpirulina promoter or under control of an exogenous promoter includingstrong promoters and inducible/repressible promoters. In an embodiment,a promoter regulating expression of crtW and crtZ genes may be a tightlyregulated promoter such that in the absence of induction there is no oressentially no expression of the exogenous genes.

For example, to produce carotenoids, a modified Spirulina may contain anoverexpressed carotenoid hydroxylase (e.g., β-carotene hydroxylase). Inthese and related embodiments, carotenoid production can be furtherincreased by subjecting the modified photosynthetic microorganism to astress condition such as, but not limited to, nitrogen deprivation. Oneillustrative carotenoid hydroxylase is encoded by crtR of A. plantensisNIES39_R00430. Another illustrative carotenoid hydroxylase is encoded bycrtZ of Pantoea ananatis. Also included are homologs or paralogsthereof, functional equivalents thereof, and fragments or variantsthereofs. Functional equivalents can include carotenoid hydroxylase withthe ability to add hydroxyl groups to β-carotene. These and relatedembodiments can be further combined with reduced expression and/oractivity of an endogenous gylcogen-pathway gene (e.g., g/gC in A.plantensis), described herein, to shunt carbon away from glycogenproduction and towards carotenoids.

Modified Phycocyanin and/or Phycoerythrin Production

In an embodiment Spirulina may be modified to increase production ofphycocyanin and/or phycoerythrin beyond the level produced by awild-type Spirulina grown under the same condition. Phycocyanin andphycoerythrin associated with their bilin chromophores, are naturaloccurring blue and red pigments, respectively, that are used in thecosmetic, food, and medical imaging industries. These pigment moleculescan be purified from cyanobacteria.

One distinguishing feature of many cyanobacteria is a massive lightharvesting structure called the phycobilisome. The phycobilisome, whichgive cyanobacteria their diversity of colors, accounts for up to 60% ofthe cell's protein. These massive protein structures transfer energydirectly to the photosynthetic reaction centers for photochemistry.

The pigment molecules in phycobilisomes that absorb light forphotochemistry are linear teterapyrrole molecules called bilins. Allbilins are covalently bound through a cysteine thioether linkage tophycobiliproteins. There are three major classes of phycobiliproteins inphycobilisomes; allophycocyanin (AP), phycocyanin (PC) and phycoerythrin(PE). Each class contains a and (3 subunits that form heterodimers. Boththe a and (3 subunits of the heterodimer can bind one or more of thebilin pigments.

The major structural components that comprise the phycobilisome can besubdivided into two groups; the “core” substructure and the “rod”substructure. In Synechococcus sp. strains, the core substructureconsists of two cylinders made of a few types of phycobiliproteins andcore linker proteins. The rod substructures are composed of hexamers ofthe PC α and PC β heterodimer. An average of three of theseheterohexamers are connected via the linker proteins, and stacked on thecore substructure.

The techniques described above may be used to add genes to Spirulinathrough targeted, homologous recombination. The techniques described inthis disclosure provide a way to add genes known to encode phycocyanin,or phycoerythrin. The techniques described above may also be used to addportions of genes or polynucleotide sequences encoding polypeptidesequences that differ from wild-type phycocyanin, or phycoerythrin, butthat still have the ability to associate with a bilin chromophore.

EXAMPLES

Certain embodiments of the present disclosure now will be illustrated bythe following Examples. The present disclosure may, however, be embodiedin many different forms and should not be construed as limited to theembodiments set forth herein; rather, these embodiments are provided sothat this disclosure will be thorough and complete, and will fullyconvey the scope of the disclosure to those skilled in the art.

Example 1

Athrospira plantensis NIES-39 cells obtained from the Microbial CultureCollection at the National Institute for Environmental Studies (Japan)were cultured in SOT medium with shaking under continuous illumination.When the optical density at 750 nm reached 0.6-0.8, cells were harvestedat 1,500×g for 10 min from 60 mL of culture and resuspended in 30 mL ofa pH balancer and salt solution. Cells were collected by centrifugationunder the same conditions and washed three times with a polyetherosmotic stabilizer. The cell suspension was twice diluted in the osmoticstabilizer and then spun down at 1,500×g for 5 min and a total volume ofthe cell suspension was adjusted to 2 mL.

FIG. 1 shows a targeted gene locus of A. plantensis. This gene locusincludes the pilA gene (encoding pili protein). Regions of the A.plantensis genome corresponding to the left homology arm and the righthomology arm are also shown. The left homology arm is adjacent to the“left” end (5′-end) of the gene locus of interest and the right homologyarm is adjacent to the “right” end (3′-end) of the gene locus ofinterest. In this example the left homology arm and the right homologyarm are about 2000 bp long.

FIG. 2 shows the pApl-pilA/aadA plasmid generated by assembling four PCRfragments containing a vector backbone, the left homology arm, the righthomology arm and the aminoglycoside adenlytransferase gene (aadA) usingthe Gibson assembly method. This plasmid was then used to transform A.plantensis. The pApl-pilA/aadA plasmid includes aadA placed between thehomology arms. aadA confers resistance to spectinomycin andstreptomycin. Thus, homologous recombination with this plasmid replacesthe pilA gene with the aadA gene. The plasmid also contains the AmpRgene (ampicillin resistance) under control of an AmpR promoter and anorigin of replication (ori) in E. coli. 2.5 μg of this plasmid was mixedwith 200 μL of the cell suspension. Immediately following mixing withthe plasmid, the cells were electroporated using a Gene Pulser Xcell™Microbial System (Bio-Rad, USA).

Following electroporation cells were grown without shaking at reducedillumination in SOT medium for 48 h then transferred to SOT mediumsupplemented with streptomycin. Presence of streptomycin in the mediumselected for transformed cells. The medium including streptomycin wasreplaced every few days to maintain efficacy of the antibiotic. Aftergrowth in the streptomycin medium, the cells were cultured in SOT mediumwith streptomycin and spectinomycin.

FIG. 3A is a schematic of substitution of the open reading frame of pilAgene in A. plantensis with the aminoglycoside adenylyltransferase (aadA)gene contained in the pApl-pilA/aadA plasmid. Following successfulhomologous recombination the pilA gene was replaced with the aadA gene.Homology arms are shown as thick lines in the schematic. Three primersA, B, and C were identified that bind to the A. plantensis genome andtwo primers D and E were identified that bind to the aadA gene in theplasmid. The primers A and C were designed to initiate DNA synthesis bya DNA polymerase outside of the homology arms that exist on theaadA-containing plasmid. Thus, primers A and C bind to regions of the A.plantensis genome that remain unchanged following transformation. PrimerA has SEQ ID NO: 13. Primer C has SEQ ID NO: 14. Primer B binds to thehomology arm upstream of the pilA gene. Primer B has SEQ ID NO: 15.Primers D and E bind to the upstream and downstream ends, respectively,of the aadA gene. Primer D has SEQ ID NO: 16. Primer E has SEQ ID NO:17.

FIG. 3B shows results of polymerase chain reaction (PCR) conducted withcombinations of different primer pairs comparing wild-type A. plantensis(WT) and A. plantensis following transformation with the pApl-pilA/aadAplasmid (ΔpilA) as described above. Supernatants of boiled bacteriaharvested after growth in streptomycin and spectinomycin were used asPCR templates. PCR was conducted under the following conditions: initialdenaturation at 98° C. for 1 min and 35 cycles of denaturation at 98° C.for 30 sec, annealing at 71° C.-0.1° C. per cycle for 15 sec andextension at 72° C. for 1.5 min, followed by final extension at 72° C.for 2 min then cooling down to 4° C., for a reaction mixture withprimers B and C; initial denaturation at 98° C. for 1 min, followed by35 cycles of denaturation at 98° C. for 30 sec, annealing at 66° C. for15 sec and extension at 72° C. for 1 min, followed by final extension at72° C. for 2 min and cooling down to 4° C., for reaction mixtures withthe rest of primers. The integration of aadA into the targeted locus wasconfirmed using two pairs of the primers (C/E and A/D). The ladder runon the gel has prominent bands at 5 kb, 1.5 kb, and 500 bp.Amplification using the C/E primer pair was not detected on WT but didyield a strong band at 2.2 kb on ΔpilA. Amplification with the A/Dprimer pair was similarly was not detected on WT but did yield a strongband at 2.1 kb on ΔpilA. Use of the primers B and C allows PCRamplification of the genomic sequences spanning either of pilA or aadA.Amplification of the WT sample (containing pi/A) generated a slightlyshorter PCR product of about 3.1 kb than amplification of thetransformed sample ΔpilA (containing aadA) of about 3.4 kb. Thisconfirms expectations because the aadA sequence is about 300-bp longerthan the pilA sequence. The PCR results demonstrate that the endogenouspilA gene was replaced with the aadA gene.

FIG. 3C shows that PCR with primers F and G complementary to the pilAgene failed to amplify any DNA from transformed cells grown in SOTmedium supplemented with streptomycin and spectinomycin indicating thatevery copy of the pilA gene was replaced in cultures grown in thepresence of the antibiotics. However, amplification with primers F and Gamplified a 340-bp sequence from WT grown in SOT medium withoutantibiotics confirming that primers F and G are effective at amplifyingthe pilA gene. Primer F has SEQ ID NO: 18. Primer G has SEQ ID NO: 19.The ladder run on this gel has prominent bands at 5 kb, 1.5 kb, and 500bp.

Example 2

Arthrospira sp. PCC 8005 cells were obtained from the Pasture CultureCollection (PCC) of Cyanobacteria maintained in the LaboratoryCollection of Cyanobacteria, at Pasteur Institut (Paris) and cultured inSOT medium with shaking under continuous illumination for three days.The cells were diluted by 3 or 4 times in fresh SOT medium during thethree day period. The cells were maintained under the above conditionsuntil the optical density at 750 nm reached a desired level. Cells werethen harvested at 3,200×g for 10 min from the culture. The supernatantwas removed and the cells were suspended in a pH balancer and saltsolution to neutralize pH. After centrifugation under the sameconditions, cells were washed with 1-50 mL of 0.1-100 mM NaCL and anosmotic stabilizer three times, and then transferred into two 1.5 mLpolypropylene microcentrifuge tubes (e.g., Eppendorf® tubes). The 1.5 mLmicrocentrifuge tubes were centrifuged at 3,200×g for 5 min at roomtemperature, and the supernatant was discarded. The cell suspension weretwice diluted in NaCL and osmolyte solution then centrifuged again at3,200×g for 5 min. The supernatant was removed and a total volume of thecell suspension was adjusted to 450 μl.

FIG. 4 shows the pApl-pilA8005/aadA plasmid (SEQ ID NO: 2) generated byusing the Gibson assembly method to join four PCR fragments containing avector backbone, a left homology arm, a right homology arm, and aadAencoding aminoglycoside adenlytransferase. In the pApl-pilA8005/aadAplasmid, aadA is located between the right and left homology arms whichcontain sequences identical to the genomic sequences found upstream anddownstream of the pilA open reading frame in Arthrospira sp. PCC 8005.The left homology arm is 2.0 kb and the right homology arm is 1.0 kb.aadA confers resistance to spectinomycin and streptomycin. Thus,homologous recombination between Arthrospira sp. PCC 8005 and thisplasmid replaces the pilA gene of Arthrospira sp. PCC 8005 with the aadAgene. The pApl-pilA8005/aadA plasmid also contains an operon encodingβ-lactamase (AmpR) which confers ampicillin resistance under control ofan AmpR promoter and an origin of replication (ori) from E. coli.

Four hundred μL of the suspended cells were mixed with 0.1-20 μg of thepApl-pilA8005/aadA plasmid, and transferred into an electroporationcuvette. Immediately following mixing, electroporation was performedusing a Gene Pulser Xcell™ Microbial System (Bio-Rad, USA)electroporation system.

Following electroporation, 1 mL of SOT medium was added into the cuvettethen the cells and SOT medium were transferred into a 50 mLpolypropylene conical centrifuge tube (e.g., Falcon® tube) containing 9mL of SOT medium. Cells were incubated at reduced illumination at 30° C.Successful transformants identified by antibiotic resistance wereselected in the presence of streptomycin and spectinomycin.

FIG. 5A is a schematic of substitution of the open reading frame of pilAgene in Arthrospira sp. PCC 8005 with the aminoglycosideadenylyltransferase (aadA) gene contained in the pApl-pilA8005/aadAplasmid. Following successful homologous recombination the pilA gene wasreplaced with the aadA gene. Homology arms are shown as shaded boxes inthe schematic. Two primers H and I were identified that bind to theArthrospira sp. PCC 8005 genome and one primer J was identified thatbinds to the aadA gene in the plasmid. Primer H initiates DNA synthesisby a DNA polymerase outside of the homology arms that exist on thepApl-pilA8005/aadA plasmid. Thus, primer H binds to a region of theArthrospira sp. PCC 8005 genome that remains unchanged followingtransformation. Primer H has SEQ ID NO: 20. Primer I binds to thehomology arm upstream of the pilA gene. Primer I has SEQ ID NO: 21.Primer J has SEQ ID NO: 22.

FIG. 5B shows results of PCR conducted on DNA extracted from the cellscultured in the presence of the antibiotics. The PCR was performed withprimer pairs H/I and H/J on wild-type Arthrospira sp. PCC 8005 (WT) andArthrospira sp. PCC 8005 following transformation with thepApl-pilA8005/aadA plasmid resulting in deletion of pilA (ΔpilA). PCRwas conducted under the conditions described above in Example 1. Becausethere is no annealing site for primer J in wild-type Arthrospira sp. PCC8005, the H/J primer pair will result in amplification only in thereaction mixtures containing DNA extracted from the transformed cells,indicating that pilA of Arthrospira sp. PCC 8005 was replaced with aadA.The integration of aadA into the targeted locus was confirmed using twopairs of the primers (H/I and H/J) and comparing the results by runningthe PCR products on a 1% w/v agarose gel.

Amplification of DNA extracted from wild-type Arthrospira sp. PCC 8005with primer pair H/I shows a band between 2.5 kb and 3 kb indicatingpresence of the pilA gene. As expected, amplification of WT Arthrospirasp. PCC 8005 with the H/i primer pair does not result in any bandbecause without transformation aadA is not present in the Arthrospirasp. PCC 8005 genome. DNA extracted from the transformed cells resultedin PCR products when amplified with either the H/I primer pair or theH/i primer pair. When the H/I primers are used on DNA from transformedcells, a band is present at approximately 3 kb which is slightly largerthan the band resulting from H/I primer pair amplification of wild-typeArthrospira sp. PCC 8005. This demonstrates that the chromosomecontaining aadA was fully segregated since aadA is approximately 300 bplonger than pilA. The transformed cells, unlike the WT cells, alsoamplified with the H/i primer pair yielding a band slightly above 2 kb.The PCR results demonstrate that the endogenous pilA gene was replacedwith the aadA gene and that the aadA gene was duplicated on allchromosomal copies.

Example 3

Athrospira plantensis NIES-39 was modified using the procedure fromExample 2 to introduce exogenous genes encoding β-carotene3,3′-hydroxylase (CrtZ) and β-carotene 4,4′-ketolase (CrtW) into theendogenous NIES39_Q01220 locus. A. plantentis does not naturallysynthesize astaxanthin, but introduction of crtZ and crtW provides abiosynthesis pathway enabling the synthesis of this valuable carotenoid.The crtZ gene was obtained from Pantoea ananatis (SEQ ID NO: 23) and thecrtW gene was obtained from Brevundimonas sp. SD212 (SEQ ID NO: 24).Both genes have been verified to function in Synechococcus elongatusPCC7942 and were codon optimized for expression in cyanobacteria.

A pApl-NS1/Prs-crtW-crtZ plasmid was created to introduce crtZ and crtWinto A. plantentis. This plasmid includes the aadA gene and the crtZ andcrtW genes downstream of an inducible trc promoter connected to ariboswitch. FIG. 6 shows the design of the pApl-NS1/Prs-crtW-crtZplasmid (SEQ ID NO: 3). This plasmid was generated by the sametechniques described in Example 1.

Electroporation and culturing techniques were initially performed asdescribed in Example 1 but for using the pApl-NS1/Prs-crtW-crtZ plasmidinstead of the pApl-pilA/aadA plasmid from Example 1. Following thischange transformation was not successful. Without being bound by theoryexpression of CrtW and CrtZ in cyanobacteria is believed to disruptphotosystems which may inhibit cell growth. It is theorized that theselected promoter, absent induction, did not sufficiently repressexpression of the crtZ and crtWgenes. Thus, presence of CrtW and CrtZproteins may have prevented any transformed cells from growing.Transformation with genes that can inhibit growth may be dependent on apromoter design that very tightly regulates gene expression. Interactionbetween promotors, inserted genes, and transformation success is an areaof further study.

Example 4

Athrospira plantensis NIES-39 is modified by adding additional copies ofthe cpcA and cpcB genes encoding C-phycocyanin a and (3 subunits. WTAthrospira plantensis NIES-39 has only a single set of these genes.Increasing the copy number of these endogenous genes increases synthesisof blue-colored C-phycocyanin. The cpcA (SEQ ID NO: 25) and cpcB (SEQ IDNO: 26) genes were obtained from WT Athrospira plantensis NIES-39.

The plasmid used to introduce cpcB and cpcA into A. plantentis wascreated by the techniques described in Example 1. FIG. 7 shows aschematic of the pApl-NS1/aadA-cpcBA plasmid (SEQ ID NO: 4). Thisplasmid contains a low-copy p15A origin of replication, the pilApromoter (a 168-bp sequence upstream of the pilA open reading frame),aadA, and the genomic sequence (SEQ ID NO: 27) ranging from 278-bpupstream of cpcB to the 3′ end of cpcA in the middle of gene locusNIES39_Q01220. The 1.5-kb flanking regions of the insertion site are theleft and right homology arms that integrate the exogenous gene operonsinto the targeted gene locus (i.e. NIES39_Q01220).

Athrospira plantensis NIES-39 was transformed with thepApl-NS1/aadA-cpcBA plasmid using the protocol we described in Example3.

Following transformation, the strain carrying the second copies of cpcBand cpcA was grown in 160 mL of SOT medium in under the same conditionsas its parental strain (i.e. Arthrospira platensis NIES-39). Cells wereharvested from 12 mL of each culture every other day to quantify theamounts of C-phycocyanin and allophycocyanin. The collected cells wereresuspended in 5 mL of 100 mM sodium phosphate buffer (pH 6.0), andcentrifuged at 3,200×g for 5 min at room temperature. The pellets weresuspended in 2.5 mL of 100 mM sodium phosphate buffer (pH 6.0); and 700μl of the suspended cells were transferred on pre-scaled PVDF(polyvinylidene fluoride or polyvinylidene difluoride) membranes. Thecells were extensively washed with water (15-20 mL×2) on the membranes,and dried in an oven overnight in order to calculate the weight of drybiomass. The remaining cells suspended in 100 mM sodium phosphate buffer(pH 6.0) were stored at −80° C. and diluted in the same 100 mM sodiumphosphate buffer to 2.0 mg dry cell weight per mL of buffer. The cellswere lysed by gentle agitation at 30° C. overnight and centrifuged at16,000×g for 30 min at 4° C. The supernatant was transferred into newtubes, and centrifuged again under the same conditions. The supernatantwas diluted in 100 mM sodium phosphate buffer (pH 6.0) and the opticaldensities were measured between 450 nm and 750 nm in 5-nm intervals toconfirm that chlorophyll and carotenoids were excluded. The contents ofC-phycocyanin (Cpc) and allophycocyanin (Apc) were calculated using theoptical densities of clarified cell lysates at 620 nm and 650 nm.Concentrations of C-phycocyanin and allophycocyanin were calculatedusing the equation OD_(λ)=C_(Cpc)×E_(λ),Cpc+C_(Apc)×E_(λ,Apc) in which Aequals 620 or 650 nm, where C_(Cpc) is the concentration ofC-phycocyanin (mg/mL), E_(λ,Cpc) is the extinction coefficient ofC-phycocyanin, C_(Apc) is the concentration of allophycocyanin (mg/mL),E_(λ,Apc) is the extinction coefficient of allophycocyanin, and OD(optical density) is the absorbance. Use of this equation is describedin Yoshikawa and Belay (2008) J. AOAC Int.

FIGS. 8A-D are graphs comparing phycocyanin production and growth ofArthrospira platensis NIES-39 transformed to include a total of twocopies of each of cpcA and cpcB (dotted lines) and WT Arthrospiraplatensis NIES-39 (solid lines). Error bars for all graphs show standarddeviation from three independent experiments. Cells were harvested fromthe cultures every other day i.e. on days 2, 4, and 6 as describedabove. The data for all FIGS. 8A-D were gathered from cells grown underthe same conditions.

FIG. 8A shows Cpc and Apc production measured as percent of dry biomass.The transformed strain (dotted line) had approximately 3-5% more of itsbiomass as Cpc than the WT strain (solid line). For both strains thepercentage of Cpc peaked on day 4 then decrease. Levels of Apc wereabout 1% lower in the transformed strain than in the WT strain.

Table 1 shows phycocyanin levels as a percentage of dry biomass furtheranalyzed as pure phycocyanin and crude phycocyanin.

TABLE 1 % dry biomass pApI-NS1/aadA-cpcBA Day WT transformed strain PurePhycocyanin (%) 2 8.68 ± 0.372 12.2 ± 0.490 4 12.0 ± 0.406 16.3 ± 0.6856 9.96 ± 0.476 13.7 ± 0.391 Crude Phycocyanin (%) 2 18.6 ± 0.802 26.3 ±1.06 4 25.8 ± 0.875 35.1 ± 1.48 6 21.4 ± 1.02 29.6 ± 0.843

Pure phycocyanin was calculated using the equation:

${\% \mspace{14mu} {of}\mspace{14mu} {dry}\mspace{14mu} {bio}\mspace{14mu} {mass}\mspace{14mu} {as}\mspace{14mu} {pure}\mspace{14mu} {phycocyanin}} = {100{\left( \frac{{OD}_{260} \times {dilution}\mspace{14mu} {factor}}{7.3 \times {dry}\mspace{14mu} {weight}} \right).}}$

Crude phycocyanin represents all phycobiliprotein pigments and wascalculated using the equation:

${\% \mspace{14mu} {of}\mspace{14mu} {dry}\mspace{14mu} {bio}\mspace{14mu} {mass}\mspace{14mu} {as}\mspace{14mu} {crude}\mspace{14mu} {phycocyanin}} = {100{\left( \frac{{OD}_{260} \times {dilution}\mspace{14mu} {factor}}{3.39 \times {dry}\mspace{14mu} {weight}} \right).}}$

FIG. 8B shows Cpc and Apc production measured as percent of totalsoluble proteins. For both the WT (solid line) and transformed (dottedline) strains the percent of Cpc increased with time. The transformedstrain had approximately 5% more of it total soluble proteins as Cpcthan the WT strain. Levels of Apc were lower and relatively stable forboth strains with the transformed strain having about 1% fewer of totalsoluble proteins as Apc than the WT strain.

FIG. 8C shows total Cpc and Apc production in grams of phycocyanin perliter of cell culture. The total Cpc production of the transformedstrain (dotted line) was more than that of the WT strain (solid line) atall time points and the difference increased with time. Apc levels alsoincreased with time, but the WT strain produced higher levels of Apcthan the transformed strain. The contents of Cpc and Apc varieddepending on the growth phase, but at all time points and under allconditions, the transformed strain accumulated 50-100% greater amountsof Cpc and less Apc than the WT strain.

FIG. 8D compares growth of the WT and transformed strains. Thetransformed strain grew (dotted line) at the same rate as the WT strain(solid line) on days 0-4 and only slightly slower on day 6.

Example 5

To analyze extracts from WT Arthrospira platensis NIES-39 and thetransformed strain described above, both strains were cultured in 50 Lphotobioreactors of SOT medium to obtain biomass enough for proteinextraction. The cultures were concentrated using a filter cartridge thenthe cells were harvested by centrifugation at 3,000×g for 10 min. Theconcentrated cells were then lyophilized. Two grams of dried biomassfrom each of the two strains were suspended in 20 mL of water andcentrifuged at 3,200×g for 5 min to collect swelling biomass. Thesupernatant was removed and the pellet was suspended in 20 mL of water.After centrifugation under the same conditions, the pellets were thensuspended in 80 mL of 20 mM sodium phosphate buffer (pH 6.0) by repeatedpipetting and gently stirred at room temperature for 30 min. Sonicationwas performed twice at 50% of the maximal amplitude for 30 sec at roomtemperature using a Qsonica sonicator Q700. Following sonication, thesuspended cells were stirred continually overnight at room temperature.To recover water-soluble extracts from the cell lysates, the sampleswere centrifuged at 4° C. for 30 min at 10,000×g once, then at 16,000×gtwice. Seventy two milliliter of the supernatant from each cell lysatewas transferred into a glass beaker, and 45 mL of 100% saturatedammonium sulfate in 20 mM sodium phosphate (pH 6.5) was graduallydropped into the beaker where the clarified cell extract was gentlystirred at 4° C. The mixtures of the cell lysates and ammonium sulfatewere stirred for an hour, precipitated proteins were recovered bycentrifugation at 16,000×g for 30 min at 4° C. The pellet was suspendedwith 20 mM sodium phosphate buffer (pH 6.0) to adjust the total volumeto 50 ml, and dialyzed using Slide-A-Lyzer cassettes against 2 L of 20mM sodium phosphate buffer (pH 6.0) at 4° C. The outer buffer wasexchanged three times (3 hours, overnight, then 4 hours). The sampleswere centrifuged at 4° C. for 30 min at 16,000×g, and the recoveredsupernatant were centrifuged again under the same conditions.

FIG. 9A is a photograph showing that supernatants recovered from thefinal centrifugation of the two strains have different intensity of bluecolor. The tube 900 containing WT Arthrospira platensis NIES-39 has alighter blue color; and the tube 902 containing the strain transformedwith the pApl-NS1/aadA-cpcBA plasmid has a darker blue color.C-phycocyanin has an intense blue color with a single visible absorbancemaximum around 620 nm. Thus, the deeper blue color was expected from thesupernatant of the C-phycocyanin-overproducing strain.

FIG. 9B is a graph showing absorption differences between thetransformed strain (solid line) and the WT strain (dotted line). Bothstrains exhibited an absorbance peak around 620 nm. The peak absorbancefor the transformed strain was approximately 0.60 cm⁻¹ and the peak forthe WT strain was approximately 0.35 cm⁻¹. This quantitatively confirmsthe qualitative color difference shown in FIG. 9A. Quality ofC-phycocyanin extract can be determined by the ratio of the opticaldensity at 620 nm (OD₆₂₀) to the optical density at 280 nm (OD₂₈₀). Ahigher ratio indicates a higher yield and purity of C-phycocyanin thatmay be suitable for higher-value uses such as cosmetics rather thanlower value uses such as food grade dye. The graph in FIG. 9B shows thatthe OD₆₂₀/OD₂₈₀ ratio for the transformed strain is 2.5 compared to 1.8for the WT strain. This indicates a 40% higher concentration ofC-phycocyanin from the recovered supernatant of the transformed strain.

ILLUSTRATIVE EMBODIMENTS

Embodiment 1 is a Spirulina comprising at least one stable, targetedmutation.

Embodiment 2 is the Spirulina of any one of embodiments 1, 4-11, or 25,wherein the Spirulina is Athrospiria platensis or Arthrospira maxima.

Embodiment 3 is the Spirulina of any one of embodiments 1, 2, 4-11, or25, wherein the Spirulina is Athrospiria platensis NIES-39 orArthrospira sp. PCC 8005.

Embodiment 4 is the Spirulina of any one of embodiments 1-3, 5-11, or25, wherein the stable targeted mutation is inherited for at least 10generations.

Embodiment 5 is the Spirulina of any one of embodiments 1-4, 6-11, or25, wherein the stable targeted mutation is integrated into a chromosomeof the Spirulina.

Embodiment 6 is the Spirulina of any one of embodiments 1-5, 7-11, or25, wherein the targeted mutation comprises a deletion or disruption ofat least a portion of a gene.

Embodiment 7 is the Spirulina of any one of embodiments 1-6 or 8-11wherein the targeted mutation comprises an addition of an additionalcopy of an endogenous gene or addition of an exogenous gene.

Embodiment 8 is the Spirulina of any one of embodiments 1-7, 10, or 11wherein the targeted mutation comprises addition of a gene regulatoryelement.

Embodiment 9 is the Spirulina of embodiment 8, wherein the generegulatory element is a promoter, a regulatory protein binding site, aRNA binding site, a ribosome binding site, or a RNA stability element.

Embodiment 10 is the Spirulina of any one of embodiments 1-9 or 11wherein the targeted mutation comprises addition of an exogenous orendogenous protein domain.

Embodiment 11 is the Spirulina of embodiment 10, wherein the exogenousprotein domain is a phosphorylation site, a stability domain, a proteintargeting domain, or protein-protein interaction domain.

Embodiment 12 is a method of creating a targeted mutation in Spirulina,the method comprising: contacting the Spirulina with an osmoticstabilizer; contacting the Spirulina with a vector having homology arms;and inducing artificial competence in the Spirulina.

Embodiment 13 is the method of any one of embodiments 12 or 15-24,wherein the Spirulina is Spirulina platensis.

Embodiment 14 is the method of any one of embodiments 12 or 15-24,wherein the Spirulina is Athrospiria platensis NIES-39 or Arthrospirasp. PCC 8005.

Embodiment 15 is the method of any one of embodiments 12-24, wherein theosmotic stabilizer is at least one of polyethylene glycol, ethyleneglycol, glycerol, glucose, or sucrose.

Embodiment 16 is the method of any one of embodiments 12-24, wherein thevector is a DNA vector, a linear vector, a circular vector, a singlestranded polynucleotide, or a double stranded polynucleotide.

Embodiment 17 is the method of any one of embodiments 12-24, wherein thevector comprises a vector having SEQ ID NO: 1, SEQ ID NO: 2, or SEQ IDNO: 4.

Embodiment 18 is the method of any one of embodiments 12-24, wherein atleast one of the homology arms is at least 500 bp.

Embodiment 19 is the method of any one of embodiments 12-24, wherein atleast one of the homology arms is at least 1000 bp.

Embodiment 20 is the method of any one of embodiments 12-24, wherein atleast one of the homology arms is at least 2000 bp.

Embodiment 21 is the method of any one of embodiments 12-20 and 22-24,wherein the artificial competence is induced by incubation in a solutioncontaining divalent cations, by electroporation, or by ultrasound.

Embodiment 22 is the method of any one of embodiments 12-24, furthercomprising contacting the Spirulina with a pH balancer.

Embodiment 23 is the method according to any one of embodiments 22 or24, wherein contacting the Spirulina with the pH balancer is prior tocontacting the Spirulina with the vector.

Embodiment 24 is the method according to any one of embodiments 22 or23, wherein a pH of a media containing the Spirulina and the pH balanceris about 7 to about 8.

Embodiment 25 is a Spirulina lacking at least one protein as a result ofintroducing a modification at the loci of the protein by transformationof the Spirulina with at least one DNA construct comprising a sequencehomologous with at least a portion of the loci and the modification andintegration of the DNA construct at the loci, the Spirulina beingotherwise capable of functioning in its native manner.

Embodiment 26 is a Spirulina comprising an exogenous gene encodingphycocyanin as a result of introducing the exogenous gene at a targetedloci by transformation of the Spirulina with at least one DNA constructcomprising a sequence homologous with at least a portion of the targetedloci and the exogenous gene and integration of the DNA construct at thelocus, the Spirulina producing an increased amount of phycocyaninrelative to wild-type Spirulina and maintaining the transformationthroughout multiple generations.

What is claimed is:
 1. A method for producing a phycobiliprotein, comprising: a) culturing a stable transformant Arthrospira cell comprising an introduced targeted nucleotide mutation incorporated into the cell's genome, under conditions suitable for the production of the phycobiliprotein, wherein the introduced targeted nucleotide mutation is adjacent to a region of nucleotides that is homologous to a nucleotide homology arm contained in a vector utilized to transform said cell, and wherein the stable transformant Arthrospira cell produces an increased amount of the phycobiliprotein relative to a wild-type Arthrospira cell.
 2. The method according to claim 1, wherein the phycobiliprotein is allophycocyanin.
 3. The method according to claim 1, wherein the phycobiliprotein is phycocyanin.
 4. The method according to claim 1, wherein the phycobiliprotein is C-phycocyanin.
 5. The method according to claim 1, wherein the phycobiliprotein is phycoerythrin.
 6. The method according to claim 1, wherein the cell is an Arthrospira platensis cell or Arthrospira maxima cell.
 7. The method according to claim 1, wherein the introduced targeted nucleotide mutation is stable for at least 50 generations.
 8. The method according to claim 1, wherein the introduced targeted nucleotide mutation comprises a deletion, or disruption, of at least a portion of a gene.
 9. The method according to claim 1, wherein the introduced targeted nucleotide mutation comprises addition of an additional copy of an endogenous gene.
 10. The method according to claim 1, wherein the introduced targeted nucleotide mutation comprises addition of an exogenous gene.
 11. The method according to claim 1, wherein the introduced targeted nucleotide mutation comprises addition of a gene regulatory element.
 12. The method according to claim 1, wherein the introduced targeted nucleotide mutation comprises addition of a gene regulatory element selected from the group consisting of: a promoter, a regulatory protein binding site, a RNA binding site, a ribosome binding site, or an RNA stability element.
 13. The method according to claim 1, wherein the introduced targeted nucleotide mutation comprises addition of a polynucleotide sequence encoding an exogenous protein domain.
 14. The method according to claim 1, wherein the introduced targeted nucleotide mutation comprises addition of a polynucleotide sequence encoding an exogenous protein domain selected from the group consisting of: a phosphorylation site, a stability domain, a protein targeting domain, or a protein-protein interaction domain.
 15. The method according to claim 1, further comprising: recovering the phycobiliprotein.
 16. A method for producing a product of interest, comprising: a) culturing a stable transformant Arthrospira cell comprising an introduced targeted nucleotide mutation incorporated into the cell's genome, under conditions suitable for the production of the product of interest, wherein the introduced targeted nucleotide mutation is adjacent to a region of nucleotides that is homologous to a nucleotide homology arm contained in a vector utilized to transform said cell, and wherein the stable transformant Arthrospira cell produces an increased amount of the product of interest relative to a wild-type Arthrospira cell.
 17. The method according to claim 16, wherein the product of interest is at least one selected from the group consisting of: lipid, neutral lipid, wax ester, triglyceride, triacylglycerol, biofuel, fatty acid, fatty acid alkyl ester, glycerin, enzyme, alcohol, fatty alcohol, glycogen, pigment, carotenoid, peptide, polypeptide, and protein.
 18. The method according to claim 16, wherein the product of interest is a peptide.
 19. The method according to claim 16, wherein the product of interest is a polypeptide.
 20. The method according to claim 16, wherein the product of interest is a protein.
 21. The method according to claim 16, wherein the product of interest is a phycobiliprotein.
 22. The method according to claim 16, wherein the product of interest is a phycobiliprotein selected from the group consisting of: allophycocyanin, phycocyanin, C-phycocyanin, and/or phycoerythrin.
 23. The method according to claim 16, wherein the cell is an Arthrospira platensis cell or Arthrospira maxima cell.
 24. The method according to claim 16, wherein the introduced targeted nucleotide mutation is stable for at least 50 generations.
 25. The method according to claim 16, wherein the introduced targeted nucleotide mutation comprises a deletion, or disruption, of at least a portion of a gene.
 26. The method according to claim 16, wherein the introduced targeted nucleotide mutation comprises addition of an additional copy of an endogenous gene.
 27. The method according to claim 16, wherein the introduced targeted nucleotide mutation comprises addition of an exogenous gene.
 28. The method according to claim 16, wherein the introduced targeted nucleotide mutation comprises addition of a gene regulatory element.
 29. The method according to claim 16, wherein the introduced targeted nucleotide mutation comprises addition of a gene regulatory element selected from the group consisting of: a promoter, a regulatory protein binding site, a RNA binding site, a ribosome binding site, or an RNA stability element.
 30. The method according to claim 16, further comprising: recovering the product of interest. 